To identify lead candidates for drug discovery programs, large numbers of compounds are often screened for their activities as enzyme inhibitors or receptor agonists or antagonists. Increased numbers of compounds as part of combinatorial libraries can be screened using microwell plates that allow miniaturization of assays. High throughput screening (HTS) has provided the ability to screen potential drug candidates rapidly on a miniaturized scale as described in pending U.S. patent application Ser. No. 08/868,280, filed Jun. 3, 1997. HTS involving miniaturized microwell plates (1 .mu.L/well) requires both accurate sample handling and sensitive assay methods as described in international patent application Ser. No. PCT/US98/00494, filed Jan. 8, 1998. Typical assay methods involving biological systems and reporter genes require growth conditions for cells that promote reporter gene expression (usually as a reporter enzyme), which can involve specific media and/or an inducer. A further requirement is addition of a necessary substrate for the reporter enzyme, in order to detect a measurable response related to enzyme activity in the presence or absence of an activator or suppressor of the applicable cellular pathway. Currently, each measurement requires a subsequent addition of substrate. Repeated or serial additions increase both the chances for mistakes and the propagated error, and preclude replicate measurements when `irreversible steps` (e.g., cell lysis) are needed. These multistep methods for HTS represent a disadvantage in achieving a streamlined, efficient assay for evaluating potential drug candidates.
New targets for therapeutic discovery are emerging from current research in human genetics and eukaryotic molecular biology, which enable the identification and understanding of factors that control the level of gene expression at transcriptional and post-transcriptional levels. Humans and other eukaryotic organisms have developed control mechanisms for biological pathways that achieve rapid and concerted responses to environmental stimuli. Often, these control mechanisms exert their influence at the genetic level, by controlling the expression levels of particular genes. The use of reporter genes constitutes an approach to quantitative determination of gene expression. Reporter genes are often coupled to a promoter, enhancer, or other sequence of interest; i.e. two genetic elements including a specifically measurable coding region are fused together as an artificial construct, which is then introduced into the eukaryotic cells. By measuring the level of reporter gene expression from this construct, indirect determination of the effect of the various experimental conditions, including activation or suppression of a receptor, is then possible. By inference, compounds that influence the level of reporter gene expression affect the control mechanisms in whole animal models and human clinical candidates and, therefore, have therapeutic potential.
Conventional reporter genes include those expressing E. coli .beta.-galactosidase [.beta.-gal] (An, G., Hidaka, K., Siminovitch, L., Mol. Cell. Biol. 2, 1628-1632 (1982)), xanthine-guanine phosphoribosyl transferase (Chu, G., Berg, P., Nucleic Acid Res. 13, 2921-2930 (1985)), galactokinase (Schumperli, D., Howard, B., Rosenberg, M., Proc. Natl. Acad. Sci. USA 79, 257-261 (1982)), secreted .beta.-lactamase (Cartwright, C. P., Li, Y., Zhu, Y. S., Kang, Y. S., Tipper, D. J., Yeast 10, 497-508 (1994)), .beta.-tactamase expressed intracellularly (Zlokarnik, G., Negulescu, P. A., Knapp, T. E., Mere, L., Burres, N., Feng, L., Whitney, M., Roemer, K., and Tsien, R. Y., Science 279(5347), 84-88 (1998)), thymidine kinase (Searle, P., Stuart, G., Palmiter, R., Mol. Cell. Biol. 5, 1480-1489 (1985)), chloramphenicol acetyltransferase (Gorman, C., Moffat, L., Howard, B., Mol. Cell. Biol. 2, 1044-1051 (1982)), secreted alkaline phosphatase (Berger, J., Hauber, J., Hauber, R., Geiger, R., Cullen, B., Gene 66, 1-10 (1988); Cullen, B., Malin, M., Methods Enzymol. 216, 362-368 (1992); Bronstein, I., BioTechniques 17, 172-178, (1994)), urokinase-plasminogen activator (Yokoyama-Kobayashi, M., Sugano, S., Kato, T., Kato, S., Gene 163, 193-196 (1995), Zimmerman, M., Quigley, J. P., Ashe, B., Dorn, C., Goldfarb, R., Troll, W., Proc. Natl. Acad. Sci. USA 75, 750-753 (1978), Huseby, R. M., et al., Thrombosis Research 10, 679 (1977)) and luciferase, which naturally produces a chemiluminescence signal via oxidation of organic molecules such as firefly luciferin (Structure A) (Ow, D. W., Wood, K. V., DeLuca, M.; DeWet, J. R., Helenski, D. R., Howell, S. H., Science 234, 856-859, (1986); Ow, D. W., Jacobs, J. D., Howell, S. H., Proc. Natl. Acad. Sci. USA 84, 4870-4874, (1987)). ##STR2##
From among the foregoing list of reporter genes, the gene expressing alkaline phosphatase is a particularly attractive reporter gene, in that its expression can be assayed by a simple, quick calorimetric assay for the product protein. A secreted form of human placental alkaline phosphatase, SEAP, has been readily and accurately quantified within the media of transfected cultures using either a standard calorimetric assay or a bioluminescence-based alkaline phosphatase assay. The SEAP assay has the additional advantage that multiple quantitative assays can be obtained from a single cell culture. The ability to measure levels of secretable alkaline phosphatase from cultured mammalian cells provides a universal method for the indirect determination of factors that influence gene expression (EP 327,960, Aug. 16, 1989). Measurement of SEAP in the presence of an activator or suppressor and under control conditions enables identification of potential drug candidates by their effect on cellular functions. However, SEAP suffers the disadvantage of requiring multiple step additions of substrate to each well at a controlled time to measure the activity of the enzyme within a certain time period.
The gene expressing chloramphenicol acetyltransferase [CAT] is another example of a reporter gene. It has the advantages of high protein stability, high sensitivity, and the absence of interfering activities in eukaryotic cells. Disadvantages of a CAT assay include the necessary harvesting, cell lysis, and extraction of cell cultures prior to a time-consuming assay using an expensive radioactive substrate. Assays based upon SEAP and CAT reporter genes afford results that are qualitatively and quantitatively comparable.
Reporter gene assays are typically used to evaluate agonist (stimulatory) or antagonist (inhibitory) activity of a test compound. For agonist assays, cells that conditionally express the reporter enzyme in the presence of a stimulatory signal are probed. For example, activation or stimulation of the vasoactive intestinal peptide (VIP) receptor in the human embryonic kidney cell line HEK293 is performed by assembling an artificial reporter gene construct wherein the expression of luciferase is induced by the elevation of the cyclic AMP (cAMP) levels by virtue of a genetic response element (CRE) positioned at the 5'-end of the gene. Among its many known intracellular targets, cAMP is known to activate the expression of genes that contain cAMP response elements (CRE) in their promoter/enhancer regions. Stimulation of the VIP receptor is known to cause elevation of the cAMP levels, and consequently, the HEK293(CRE-luc) cell line thus constructed responds to VIP or VIP receptor agonists by increasing the level of luciferase expression. The level of luciferase expression is determined using well-established methods (e.g., LucLite kit, available from Promega, Madison, Wis.).
The human embryonic kidney cell line HEK293 cell line can then be used to screen for VIP agonists as follows: 1) The HEK293(CRE-luc) cell line is grown in a tissue culture dish, such as a 96-well microtiter plate suitably treated for tissue culture work 2) Once the cells have adhered to the microwell surfaces, test compounds are added and the cells continue to grow for 4 to 48 hours in the presence of cell growth medium. The cell growth medium allows for the production of new proteins, and consequently any stimulation of the VIP receptor or of the endogenous pathway leading to gene expression will result in the increased production of luciferase 3) After this incubation period, the medium is removed and the luciferase assay reagent comprised of luciferin, Mg.sup.+2, and ATP, along with cell lysis reagents, is added. The activity of luciferase is then detected by the chemiluminescence of the proximal oxyluciferin product of the enzymatic reaction. By quantifying the amount of light produced, the amount of luciferase can be compared from one sample to the next and consequently the amount of stimulation of the VIP receptor pathway can be gauged. VIP agonists will therefore appear as brighter wells on an otherwise dimmer background.
For antagonist assays, the steps are similar except that a common activator (in the instance above, VIP) is initially used to bring all wells of the screening assay to a uniform brightness level in the absence of test compound. Compounds are then tested for their ability to decrease the amount of light produced from the luciferase reaction. These two classes of assay can be summarized in the following scheme: ##STR3##
Following the above scheme, cells capable of expressing a reporter enzyme and a test compound are contacted and cells are grown under conditions to encourage expression of a reporter enzyme. In an antagonist assay, a known activator is also added with the cells and test compound to activate the reporter enzyme expression. Cell lysis is required to release the reporter enzyme. Following addition of the enzyme substrate to the media, interaction of the reporter enzyme and the added substrate occurs to produce a signal proportional to the expression of the reporter enzyme in the presence of a test compound. Note that the addition of substrate is done by adding another solution to the cells. This fluid addition step is a disadvantage for cell-based assays because addition of more solution can perturb the cells and dilute the sample so as to preclude a second measurement.
Chemiluminescent detection of reporter gene expression provides flexibility and sensitivity in assays. Chemiluminescent substrates for alkaline phosphatase, such as spiroadamantane-dioxetane compounds allow highly sensitive nucleic acid analysis techniques (During, K., J. Chrom. 618, 105-131(1993)). Disodium 3-(4-methoxyspiro[1,2-dioxetane-3,2'-(5'-chloro)tricyclo[3.3.1.1.sup.3,7 ]decan]-4-yl)phenyl phosphate (CSPD) and disodium 3-(4-methoxyspiro[1,2-dioxetane-3,2'-tricyclo[3.3.1.1.sup.3,7 ]decan]-4-yl)phenyl phosphate (AMPPD) are adamantyl 1,2-dioxetane systems that provide important features for chemiluminescent substrates for alkaline phosphatase or alkaline phosphatase conjugates (Bronstein, I., J. Biolum. Cherilum. 4, 99-111(1989)). In these compounds, the energy source for chemiluminescence is the dioxetane ring; chemical stability of the substrate is provided both by the adamantyl functionality and the phosphate group. The light emission capabilities of the enzyme substrate are defined by the aryl functionality. The activation site for the substrate consists of an enzyme cleavable group, in this case the phosphate ester bond. In general, the enzyme cleavable group imparts a high stability to the system, such that luminescence occurs only when the cleavable group has been removed. In this example, the arylphosphate is dephosphorylated to afford an aryloxide anion. The 1,2-dioxetane aryloxide anion, having a "weak" oxygen-oxygen bond, decomposes to form carbonyl products, and the decomposition releases approximately 400 kJ/mol of energy, resulting in a strong chemiluminescent signal.
For automated assays, a short half-life (t.sub.1/2) of the dioxetane and a short time required to reach maximum light emission for the reporter molecule are both desirable characteristics. Because the signal from a previous assay must dissipate before a second measurement can be taken, a long half-life lengthens the window between measurements, thus increasing the total time for assay and decreasing efficiency. A longer time for reaching maximum light signal emission also increases overall assay time and decreases reliability of signal readings. Long half-life and an increased time to reach maximum signal emission are disadvantages in high throughput screening assays. Chemiluminescent dioxetane derivatives that have reduced half-lives are described in U.S. Pat. No. 5,538,847, issued Jul. 23, 1996.
Typically, the more popular forms of 1,2-dioxetanes, AMPPD and CSPD (shown below) are incubated with alkaline phosphatase for determined periods of time. ##STR4## CSPD usually achieves maximum light emission at a faster rate than AMPPD when used on nylon membranes in Western blot analysis. (Albrecht, S., et al, "Bioluminescence and Chemiluminescence: Current Status", Stanley, P., Kricka, L.(Eds.), 115-118, (1991)). Reaction of AMPPD with alkaline phosphatase is outlined in Scheme 1.
Scheme 1. Use of Representative SEAP substrate (AMPPD) in measuring SEAP activity in vivo. ##STR5## Dephosphorylation of the arylphosphate results in a moderately stable anion (AMPD) which decomposes to an adamantanone and methyl m-oxybenzoate, (as a charge-transfer excited species). The excited state of methyl m-oxybenzoate anion emits light at a wavelength of approximately 477 nm. Decomposition of methyl m-oxybenzoate anion results in production of chemiluminescent signal emitted in the form of a glow, with breakdown of the intermediate anion determined by the environment (e.g., the membrane used for blot assay). Exposure time must be optimized for each general assay system to obtain appropriate signals. Generally, CSPD is preferred over AMPPD when longer emission times are needed for assays.
Chemiluminescent detection of SEAP is known. Optimum levels of SEAP in the culture medium usually are obtained 12-24 h after transfection. The levels of SEAP activity detected in the culture medium are directly proportional to changes in intracellular concentrations of its SEAP MRNA. Because the preparation of cell lysates is not necessary to assay for the reporter enzyme and the transfected cell cultures remain intact, repetitive sampling of the culture medium is possible. The assay is adaptable to automation using cultures grown in 96-microwell plates. Endogenous alkaline phosphatase activity can be eliminated by heating the culture to 65.degree. C., and the heat-stability of SEAP allows measurement of expressed SEAP only. Detection of SEAP activity from reporter gene expression requires several fluid manipulation steps involving removal of samples, dilution with buffer, heating and incubation. Addition of the chemiluminescent substrate CSPD and enhancer, followed by incubation, produces a stable chemiluminescent signal in approximately 10 minutes, and the signal remains constant for approximately one hour. However, multiple fluid manipulations, involving serial additions of the chemiluminescent substrate and the enhancer, increase the likelihood of error in measurement, particularly since maximum signal may occur before fluid addition is completed. In such a case, the maximum signal will not be obtained because the time required for fluid manipulations is too long.
A typical assay using secreted placental alkaline phosphatase (SEAP) would be designed by first assembling an artificial reporter gene construct wherein the expression of SEAP reporter gene is induced by the elevation of the cyclic AMP (cAMP) levels by virtue of a genetic response element (CRE), and then measuring the concentration of SEAP released into the medium. The HEK293(CRE-SEAP) cell line thus constructed again responds to VIP or VIP receptor agonists by increasing the level of SEAP expression. Theoretically, the reporter activity could be measured repetitively, since the cells are not destroyed by the measurement of the luminescent signal. However, repeated fluid addition to the same well is not advantageous for screening because multiple fluid additions: (a) increase error levels due to propagation of volumetric error into the overall measurement; and (b) can cause small wells to overflow as assay volumes increase with repetitive addition steps.
Chemiluminescent detection of peptidases is similar to chemiluminescent detection of SEAP and has been demonstrated with several available substrates. One commercially available substrate, CBZ-Gly-Gly-L-Arg-7-amino-4-methylcoumarin, available from Enzyme Systems Products, AMC-056, (Dublin, Calif.) can be used for the assay of urokinase-plasminogen activator. Interaction of the peptidase with the enzyme substrate results in the release of the fluorophore which can be detected as a fluorescent signal. An advantage of using a gene expressing a peptidase as a reporter gene is that the substrates are generally biocompatible and soluble under assay conditions. Another advantage arises from the fact that peptidases are usually secreted in their native environment and are stable extracellularly. Again, the disadvantage of using a peptidase as a reporter gene is that addition of the substrate is required prior to each measurement, thus introducing multiple fluid additions.
The unavailability of methods that enable assay of multiple drug candidates on a miniaturized, microvolume scale has hampered the advancement of drug discovery using high throughput screening methods. Rapid, effective means for identification of compounds that interact with eukaryotic systems to alter reporter gene expression would enhance the value of HTS in identifying potential drug candidates. For HTS applications, minimizing the number of fluid handling steps would improve accuracy and allow for facile miniaturization. It would therefore be desirable to have an enzyme substrate in the assay mixture at the initiation of the assay, but in a form that could not function as a substrate until that function was needed.